Abscisic acid mediates the reduction of petunia flower size at elevated temperatures due to reduced cell division.

MAIN CONCLUSION: Elevated temperatures suppress cell division in developing petunia buds leading to smaller flowers, mediated by ABA. Flower size is one of the most important showy traits in determining pollinator attraction, and a central factor determining the quality of floricultural products. Whereas the adverse effects of elevated temperatures on showy traits have been described in detail, its underlining mechanisms is poorly understood. Here, we investigated the physiological mechanism responsible for the reduction of flower size in petunia under elevated temperatures. We found that the early stages of flower-bud development were most sensitive to elevated temperatures, resulting in a drastic reduction of flower diameter that was almost independent of flower load. We demonstrated that the temperature-mediated flower size reduction occurred due to a shorter growth period, and a lower rate of corolla cell division. Consistently, local application of cytokinin, a phytohormone that promotes cell division, resulted in recovery of flower dimensions when grown under elevated temperatures. Hormone analysis of temperature-inhibited flower buds revealed no significant changes in levels of cytokinin, and a specific increase of abscisic acid (ABA) levels, known to inhibit cell division. Moreover, local application of ABA on flower buds caused a reduction of flower dimensions as a result of lower levels of cell division, suggesting that ABA mediates the reduction of flower size at elevated temperatures. Taken together, our results shed light on the mechanism by which elevated temperatures decrease petunia flower size, and show that temperature-mediated reduction of flower size can be alleviated by increasing the cytokinin/ABA ratio.

Metabolic engineering of plants for artemisinin synthesis.

Artemisinin, a natural compound from Artemisia annua, is highly effective in treating drug-resistant malaria. Because chemical synthesis of this natural terpenoid is not economically feasible, its only source remains as the native plant which produces only small quantities of it, resulting in a supply that is far short of demand. Extensive efforts have been invested in metabolic engineering for the biosynthesis of artemisinin precursors in microbes. However, the production of artemisinin itself has only been achieved in plants. Since, A. annua possesses only poorly developed genetic resources for traditional breeders, molecular breeding is the best alternative. In this review, we describe the efforts taken to enhance artemisinin production in A. annua via transgenesis and advocate metabolic engineering of the complete functional artemisinin metabolic pathway in heterologous plants. In both cases, we emphasize the need to apply state-of-the-art synthetic biology approaches to ensure successful biosynthesis of the drug.

Architecture and Florogenesis in Female Cannabis sativa Plants.

The inflorescence is the main product of medical cannabis. Hundreds of specialized metabolites with potential bioactivity are produced and accumulated in the glandular trichomes that are highly abundant mainly on female inflorescences. Understanding the morphophysiological and genetic mechanisms governing flower and inflorescence development is therefore of high scientific and practical importance. However, in-depth investigations of cannabis florogenesis are limited. Cannabis producers and researchers consider long photoperiod to be "non-inductive" or "vegetative," but under these growth conditions, the development of solitary flowers and bracts in shoot internodes clearly indicates that the plant cannot be defined as vegetative or non-inductive in the classical sense. Most probably, induction of solitary flowers is age-dependent and controlled by internal signals, but not by photoperiod. Short photoperiod induces intense branching, which results in the development of a compound raceme. Each inflorescence consists of condensed branchlets with the same phytomer structure as that of the larger phytomers developed under long day. Each phytomer consists of reduced leaves, bracts, one or two solitary flowers, and an axillary shoot (or inflorescence). Therefore, the effect of short photoperiod on cannabis florogenesis is not flower induction, but rather a dramatic change in shoot apex architecture to form a compound racemose inflorescence structure. An understanding of the morphophysiological characteristics of cannabis inflorescence will lay the foundation for biotechnological and physiological applications to modify architecture and to maximize plant productivity and uniformity in medical Cannabis.

Engineering of Methylation State Specific 3xMBT Domain Using ELISA Screening.

The ε-amino group of lysine residues may be mono-, di- or tri-methylated by protein lysine methyltransferases. In the past few years it has been highly considered that methylation of both histone and non-histone proteins has fundamental role in development and progression of various human diseases. Thus, the establishment of tools to study lysine methylation that will distinguish between the different states of methylation is required to elucidate their cellular functions. The 3X malignant brain tumor domain (3XMBT) repeats of the Lethal(3)malignant brain tumor-like protein 1 (L3MBTL1) have been utilized in the past as an affinity reagent for the identification of mono- and di-methylated lysine residues on individual proteins and on a proteomic scale. Here, we have utilized the 3XMBT domain to develop an enzyme-linked immunosorbent assay (ELISA) that allows the high-throughput detection of 3XMBT binding to methylated lysines. We demonstrated that this system allows the detection of methylated peptides, methylated proteins and PKMT activity on both peptides and proteins. We also optimized the assay to detect 3XMBT binding in crude E. coli lysates which facilitated the high throughput screening of 3XMBT mutant libraries. We have utilized protein engineering tools and generated a double site saturation 3XMBT library of residues 361 and 411 that were shown before to be important for binding mono and di-methylated substrates and identified variants that can exclusively recognize only di-methylated peptides. Together, our results demonstrate a powerful new approach that will contribute to deeper understanding of lysine methylation biology and that can be utilized for the engineering of domains for specific binders of other post-translational modifications.

A continuous kinetic assay for protein and DNA methyltransferase enzymatic activities.

BACKGROUND: Methyltransferases (MTs) catalyze the S-adenosylmethionine (SAM)-dependent methylation of a wide variety of protein and DNA substrates. Methylation of lysine, arginine or cytosine regulates a variety of biological processes including transcriptional activation and gene silencing. Despite extensive studies of the cellular roles of MTs, their quantitative kinetic characterization remains challenging. In the past decade, several assays have been developed to monitor methyl transfer activity utilizing different approaches including radiolabeling, antibodies or mass-spectrometry analysis. However, each approach suffers from different limitation and no easy continuous assay for detection of MT activity exists. RESULTS: We have developed a continuous coupled assay for the general detection of MTs activity. In this assay, the formation of S-adenosylhomocysteine (SAH) product is coupled NAD(P)H oxidation through three enzyme reactions including glutamate dehydrogenase leading to absorbance changes at 340 nm. The utility and versatility of this assay is demonstrated for SET7/9 and SETD6 with peptides and full length protein substrates and for M.HaeIII with a DNA substrate. CONCLUSIONS: This study shows a simple and robust assay for the continuous monitoring of MT enzymatic activity. This assay can be used for the determination of steady-state kinetic enzymatic parameters (e.g., k cat and K M) for a wide variety of MTs and can be easily adapted for high-throughput detection of MT activity for various applications.